Rspondins as modulators of angiogenesis and vasculogenesis

ABSTRACT

The present invention relates to the use of Rspondins, particularly Rspondin2 (Rspo2) or Rspondin3 (Rspo3) or Rspondin nucleic acids, or regulators or effectors or modulators of Rspondin, e.g. Rspo2 and/or Rspo3 to promote or inhibit angiogenesis and/or vasculogenesis, respectively. The invention is based on the demonstration that Rspo3 and Rspo2 are angiogenesis promoters, and the identification of Rspo2 and 3 as positive regulators of vascular endothelial growth factor (VEGF). These results indicate a major role for Rspondins, particularly Rspo3 and/or Rspo2 in the signaling system during angiogenesis. The invention also relates to the use of regulators or effectors or modulators of Rspondin3, including agonists and antagonists, in the treatment of conditions where treatment involves inhibiting or promoting angiogenesis and/or vasculogenesis.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation Application of co-pendingapplication Ser. No. 14/453,692, filed Aug. 7, 2014, which is aDivisional of application Ser. No. 14/048,225, filed Oct. 8, 2013, nowU.S. Pat. No. 8,926,970, which is a Continuation Application ofapplication Ser. No. 13/309,193, filed Dec. 1, 2011, now U.S. Pat. No.8,580,736, which is a Divisional of National Stage application Ser. No.12/311,921, filed Apr. 16, 2009, now U.S. Pat. No. 8,088,374, whichclaims priority from PCT Application No. PCT/EP2007/009105 filed Oct.19, 2007, which in turn, claims priority from EP Application Serial No.06 022 070.4, filed Oct. 20, 2006. Applicants claim the benefits of 35U.S.C. § 120 as to the U.S. Non-Provisional applications and the PCTapplication, and priority under 35 U.S.C. § 119 as to the said EPapplication, and the entire disclosures of both applications areincorporated herein by reference in their entireties.

1. INTRODUCTION

The present invention relates to the use of Rspondin polypeptides,particularly Rspondin2 (Rspo2) or Rspondin3 (Rspo3) or Rspondin nucleicacids, or regulators or effectors or modulators of Rspondinrespectively. The invention is based on the demonstration that Rspo3 andRspo2 are angiogenesis promoters, and the identification of Rspo2 and 3as positive regulators of vascular endothelial growth factor (VEGF).These results indicate a major role for Rspondins, particularly Rspo3and/or Rspo2 in the signalling system during angiogenesis. The inventionalso relates to the use of Rspondin3 regulators or effectors ormodulators, including agonists and antagonists, in the treatment ofconditions, including cancer, by modulating angiogenesis and/orvasculogenesis.

2. BACKGROUND OF THE INVENTION

The Rspondin protein family is conserved among vertebrates and consistsof the four related members Rspondin1-4 (Rspo1-4) (Chen et al., 2002,Mol. Biol. Rep. 29, 287-292, who called Rspo3 hPWTSR; Kamata et al.,2004, Biochim. Biophys. Acta.1676, 51-62; Kazanskaya et al., 2004, Dev.Cell 7, 525-534; Kim et al., 2005, Science 309, 1256-1259; Kim et al.,2006, Cell Cycle 5, 23-26; Nam et al., 2006, J. Biol. Chem. 281,13247-13257). Human Rspo1-4 were also described as Stem Cell GrowthFactor Like Polypeptides, which are able to promote proliferation ofhematopoietic stem cells (WO 01/77169; WO 01/07611). They were alsodesignated as Futrin1-4 and identified as modulators of the Wntsignalling pathway (WO 2005/040418). The content of these documents isherein incorporated by reference and the amino acid and nucleicsequences of Rspondins 1-4 disclosed therein are specifically includedherein.

The Rspo genes encode secreted proteins which can activate Wnt/b-cateninsignalling, and Rspo2 promotes myogenesis via the Wnt/b-cateninsignalling pathway in Xenopus (Kazanskaya et al., 2004, Dev. Cell 7,525-534). Rspondin genes are widely coexpressed with Wnt genes in manyregions during embryonic development, and Rspondin expression ispositively regulated by Wnt signals (Kamata et al., 2004, Biochim.Biophys. Acta.1676, 51-62; Kazanskaya et al., 2004, Dev. Cell 7,525-534). Furthermore, it was reported that secreted human Rspo1promotes proliferation of intestinal epithelium through stabilizing ofb-catenin (Kim et al., 2005 Science 309, 1256-9). Mutation of mouseRspo3 results in embryonic lethality and induces severe defects in thedevelopment of the placenta (Aoki et al., Dev Biol. 2007 301(1):218-26).However, no effect on blood vessel development was reported in thismutant model and, in contrast to the results disclosed herein, theembryos appeared to show no sign of haemorrhage, therefore there was nosuggestion before the present invention that Rspondin, in particularRspondin 2 or 3, played a significant role in angiogenesis and/orvasculogenesis.

Angiogenesis is likely to be regulated by polypeptide growth factors.Several polypeptides with in vitro endothelial cell growth promotingactivity have been identified. Examples include acidic and basicfibroblastic growth factor, VEGF and placental growth factor.

VEGF is a key factor in vasculogenesis and angiogenesis and itssignalling pathway an important target for pharmacological intervention(Ferrara 2005, Oncology 3:11-6; Rosen 2005, Oncologist 10:382-91).

3. SUMMARY OF THE INVENTION

The present invention relates to the use of Rspondin polypeptides orRspondin nucleic acids, or regulators or effectors or modulators ofRspondin polypeptides or Rspondin nucleic acids. The invention is basedon the demonstration that Rspo3 and Rspo2 are vasculogenesis andangiogenesis promoters. Further, they induce endothelial cell growth andhave been identified as positive regulators of VEGF.

The results indicate a major role for Rspondins polypeptides,particularly Rspo2 and/or Rspo3 in the signalling system duringangiogenesis and/or vasculogenesis.

Rspondin polypeptides (e.g. Rspo2 or Rspo3), Rspondin nucleic acids, andagonists of Rspondin, are suitable in the treatment of conditionswherein said treatment involves promoting angiogenesis and/orvasculogenesis

Antagonists of Rspondin polypeptides (e.g. of Rspo2 or Rspo3) or ofRspondin nucleic acids, are suitable in the treatment of conditionswherein said treatment involves inhibiting angiogenesis and/orvasculogenesis.

The invention also relates to the use of Rspondin polypeptides, Rspondinnucleic acids and regulators or effectors or modulators of Rspondin fordiagnostic applications, particularly for the diagnosis or monitoring ofangiogenesis- and/or vasculogenesis-associated processes, conditions anddisorders.

Further, the invention refers to cells and transgenic non-human animalsexhibiting modified, e.g. increased or decreased Rspondin, particularlyRpo2 and/or Rspo3, expression.

Rspondin polypeptides and Rspondin nucleic acids and cells or transgenicanimals may be used in screening procedures in order to identify and/orcharacterize effectors of angiogenesis and/or vasculogenesis.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Rspo3 is necessary for the blood vessel cell development inXenopus tropicalis. Xenopus tropicalis embryos were injected at the 4cell stage with control or Rspo3 morpholino antisense oligonucleotides(Mo) as indicated. Embryos were fixed at tailbud stage and in situhybridization for markers of blood (α-globin, SCL, Mead et al., 1998,Development 125, 2611-2620) or forming blood vessels (msr, Devic et al.,Mech Dev. 1996; 59,129-40) was carried out. Note the expansion of bloodmarkers and inhibition of msr in Rspo3 Mo-treated embryos.

FIG. 2: Demonstration of the specificity of Rspo3 morpholino antisenseoligonucleotides. Xenopus tropicalis embryos were injected in twoventral blastomers at the 4-8 cells stage with Rspo3 morpholinoantisense oligonucleotides (Mo) with and without Xenopus laevis Rspo2mRNA as indicated. At gastrula stage (stage 10) ventral marginal zones(VMZ) were excised and cultured until sibling embryos reached stage 28.VMZs were fixed and processed for whole mount in situ hybridization forthe blood marker α-globin. Note rescue of Rspo3 Mo-induced expansion ofα-globin by Rspo2 mRNA. This rescue shows the specificity of themorpholino phenotype.

FIG. 3: Rspo3 is necessary and sufficient for promoting blood vesselcell development in Xenopus tropicalis. Xenopus tropicalis embryos wereinjected at the 4 cells stage with control or Rspo3 morpholino antisenseoligonucleotides (Mo) or Xenopus laevis Rspo2 mRNA as indicated. Atgastrula stage the ventral marginal zone was excised and cultivated inisolation until stage 28. RT-PCR analysis was carried out for theindicated marker genes. H4, histone 4 for normalization. —RT, minusreverse transcriptase control. Note that Mo inhibition of Rspo3 inhibitsblood vessel marker VEGF and msr expression and induces the bloodmarkers α-globin and SCL.

FIG. 4: Expression of. Rspo3 in vasculature of mouse embryos. In situhybridisation of Rspo3 in E 10.5 mouse embryo is shown. Arrowheads pointto expression in embryonic blood vessels.

FIG. 5: Targeted mutagenesis of murine Rspo3. (A) Genomic structure ofRspo3 and targeting vector used for homologous recombination is EScells. (B) Targeted allele before and (C) after elimination of neomycineselectable marker gene using Flp recombinase.

FIG. 6: Rspo3 mutant mice show internal bleeding. Photographs ofwild-type (wt) and Rspo3-/-embryos (mutant) mice at E10.5. Notehaemorrhages in the mutant mouse, indicative of failure of blood vesselformation.

FIG. 7: Rspo3 mutant mice show reduced blood vessel formation. Wild-type(wt) and Rspo3-/-(mutant) yolk sacs of E 10.5 embryos are shown. Notepale yolk sac in mutant.

FIG. 8: Rspo3 mutant mice lose VEGF expression. Whole mount in situhybridization for VEGF is shown in placentas of wild-type (wt) andRspo3-/-embryos (mutant) of E 9.5.

FIG. 9: Rspo2 induces angiogenesis in the chicken chorioallantoicmembrane (CAM) assay.

FIG. 10: Rspo2 induces tube formation in endothelial cells. Control orXenopus laevis Rspo2 conditioned medium was applied to human endothelialcells (HDMEC) for 5 days. Note induction of morphogenesis indicative oftube formation, as is characteristic during angiogenesis.

FIG. 11: Rspo2 induces endothelial cell growth. Control or Xenopuslaevis Rspo2 conditioned medium or 0.5 ng/ml VEGF was applied to humanendothelial cells (HUVEC) for 2 days and cell proliferation was assayedusing a commercial kit (Roche).

5. DESCRIPTION OF THE INVENTION

5.1 Definitions

As used herein the term ‘Rspondin polypeptides’ according to the presentinvention refers to members of the Rspondin family which may be derivedfrom mammalian or other vertebrate organisms. The Rspondin proteinfamily consists of the four related members Rspondin1-4 (Rspo1-4).

Preferably, the Rspondin polypeptide is a human Rspondin, e.g. humanRspondin1, 2, 3 or 4. More preferably, the Rspondin polypeptide is anRspondin2 or 3 polypeptide, particularly a human Rspondin2 or 3polypeptide. The amino acid sequences of human Rspondin polypeptides 1,2, 3 and 4 are shown in WO 2005/040418, the content of which is hereinincorporated by reference. Further examples of Rspondin polypeptides areRspondins from Xenopus, e.g. Xenopus tropicalis and Xenopus laevis orfrom Mus musculus.

Further sequences for human Rspondin nucleic acid and amino acidsequences are as follows: Human Rspondin 1 nucleic acid sequence(NM_001038633, SEQ ID NO: 16), amino acid sequence (ABA54597, SEQ ID NO:17), human Rspondin 2 nucleic acid sequence (NM_178565, SEQ ID NO: 18),amino acid sequence (NP_848660, SEQ ID NO: 19), human Rspondin 3 nucleicacid sequence (NM_032784, SEQ ID NO: 20), amino acid sequence(NP_116173, SEQ ID NO: 21), human Rspondin 4 nucleic acid sequence(NM_001029871, SEQ ID NO: 22), amino acid sequence (NP_001025042, SEQ IDNO: 23).

Rspondin polypeptides are further defined herein as polypeptides thatshow at least 40%, preferably at least 60%, more preferably at least80%, at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity at the amino acid level to the respective human Rspondinpolypeptide over its entire length (Kazanskaya et al., 2004, Dev. Cell7, 525-534). Further, Rspondin polypeptides according to the inventionare preferably characterized as having at least one biological activityselected from

-   -   i induction of angiogenesis in the CAM assay,    -   ii induction of tube formation in endothelial cells,    -   iii induction of endothelial cell growth, particularly growth of        human endothelial cells, and    -   iv induction of VEGF expression.

The term ‘polypeptide’ includes to full-length proteins, proteinaceousmolecules, fragments of proteins, fusion proteins, peptides,oligopeptides, variants, derivatives, analogs or functional equivalentsthereof.

The term ‘functionally equivalent to Rspondin’ as used herein refers toa protein which induces angiogenesis and/or VEGF expression. The geneproduct itself may contain deletions, additions or substitutions ofamino acid residues within the Rspondin, e.g. Rspo2 or Rpo3 sequence,which result in a silent change thus retaining significant signaltransducing capacity thus producing a functionally equivalent Rspondin.Such amino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipatic nature of the residues involved. For example, negativelycharged amino acids include aspartic acid and glutamic acid; positivelycharged amino acids include lysine and arginine; amino acids withuncharged polar head groups having similar hydrophilicity values includethe following: leucine, isoleucine, valine; glycine, analine;asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.

As used herein the term, ‘Rspondin nucleic acid’ refers to nucleic acidsequences that encode members of the Rspondin family and which may bederived from mammalian or other vertebrate organisms. Preferably, theRspondin nucleic encodes a human Rspondin, e.g. human Rspondin1, 2, 3 or4. More preferably, the Rspondin nucleic acid encodes an Rspondin2 or 3polypeptide, particularly it encodes a human Rspondin2 or 3 polypeptide.The nucleic acid sequences of human Rspondin 1, 2, 3 and 4 are shown inWO 2005/040418, the content of which is herein incorporated byreference. Further examples of Rspondin nucleic acids are those whichencode the Rspondins from Xenopus, e.g. Xenopus tropicalis and Xenopuslaevis or from Mus musculus.

Rspondin nucleic acids are further defined herein as molecules selectedfrom

-   -   (a) nucleic acid molecules encoding Rspondin polypeptides, e.g a        human Rspondin, particularly Rspo2 and/or Rspo3,    -   (b) nucleic acid molecules which hybridize under stringent        conditions to a nucleic acid molecule of (a) and/or a nucleic        acid molecule which is complementary thereto,    -   (c) nucleic acid molecules which encode the same polypeptide as        a nucleic acid molecule of (a) and/or (b), and    -   (d) nucleic acid molecules which encode a polypeptide which is        at least 40%, preferably at least 60%, more preferably at least        80%, and most preferably at least 90% identical to a polypeptide        encoded by a nucleic acid molecule of (a) over its entire        length.

The nucleic acid molecules may be e.g. DNA molecules or RNA molecules.

Nucleic acid molecules which may be used in accordance with theinvention may include deletions, additions or substitutions of differentnucleotide residues resulting in a sequence that encodes the same or afunctionally equivalent gene product.

As used herein, the terms ‘regulators’ or ‘effectors’ or ‘modulators’ ofRspondin polypeptides or nucleic acids are used interchangeably hereinand any of the above may be used to refer to antibodies, peptides, lowmolecular weight organic or inorganic molecules and other sources ofpotentially biologically active materials capable of modulating Rspondinpolypeptides, e.g. Rspo2 and/or Rspo3 signal transduction or capable ofmodulating Rspondin polypeptide activity or capable of modulatingRspondin expression to promote (agonists) or inhibit (antagonists)angiogenesis and/or vasculogenesis. Said regulators, effectors ormodulators can be naturally occurring or synthetically produced.

As used herein, the term ‘compound capable of binding to Rspondin’refers to a naturally occurring or synthetically produced regulator,effector or modulator of Rspondin' which interacts with an Rspondinpolypeptide. Examples of such compounds are (i) a natural partner, e.g.receptor of an Rspondin; (ii) a naturally occurring molecule which ispart of the signalling complex; and/or a naturally occurring signallingmolecule produced by other cell types; (iii) naturally occurring orsynthetically produced antibody. The term ‘compound’ is used herein inthe context of a ‘test compound’ or a ‘drug candidate compound.

As used herein the term ‘agonist of Rspondin’ refers to regulators oreffectors or modulators of Rspondin that activate the intracellularresponse of Rspondin and thus promote angiogenesis and/orvasculogenesis.

As used herein, the term ‘antagonist of Rspondin’ refers to regulatorsor effectors or modulators of Rspondin polypeptides or Rspondin nucleicacids that inhibit, decrease or prevent the intracellular response ofRspondin polypeptides or Rspondin nucleic acids and thus inhibit,decrease or prevent angiogenesis and/or vasculogenesis.

Examples of suitable antagonists are mutated forms of Rspondin, having adominant negative effect, Rspondin-binding polypeptides, e.g.anti-Rspondin antibodies including recombinant antibodies or antibodyfragments containing at least one Rspondin binding site. Furtherexamples of Rspondin antagonists are nucleic acids capable of inhibitingRspondin translation, transcription, expression and/or activity, e.g.aptamers, antisense molecules, ribozymes or nucleic acid moleculescapable of RNA interference such as siRNA molecules including nucleicacid analogs such as peptidic nucleic acids or morpholino nucleic acids.Such nucleic acids may bind to or otherwise interfere with Rspondinnucleic acids.

As used herein, the term ‘antibody’ or ‘antibodies’ includes but is notlimited to recombinant polyclonal, monoclonal, chimeric, humanized, orsingle chain antibodies or fragments thereof including Fab fragments,single chain fragments, and fragments produced by an Fab expressionlibrary. Neutralizing antibodies i.e., those which compete for the VEGFbinding site of an Rspondin are especially preferred for diagnostics andtherapeutics.

As used herein, the term ‘vasculogenesis’ refers to the formation andspreading of blood vessels.

As used herein, the term ‘angiogenesis’ relates to a process whichinvolves the vascularisation of a tissue, in particular, theproliferation, migration and infiltration of vascular endothelial cellsand the growth and the development of new capillary blood vessels.

As used herein, the term ‘treating’ or ‘treatment’ refers to anintervention performed with the intention of preventing the developmentor altering the pathology of, and thereby alleviating a disorder,disease or condition, including one or more symptoms of such disorder orcondition. Accordingly, ‘treating’ refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatinginclude those already with the disorder as well as those in which thedisorder is to be prevented. The related term ‘treatment’, as usedherein, refers to the act of treating a disorder, symptom, disease orcondition, as the term ‘treating’ is defined above.

As used herein the term “conditions where treatment involves inhibitingangiogenesis and/or vasculogenesis” specifically includes (withoutlimitation) conditions such as tumor growth, e.g. solid tumor growth andmetastatic activity, atherosclerosis, stenosis, restenosis, retinopathy,macular degeneration, psoriasis and rheumatoid arthritis.

As used herein, the term ‘Conditions where treatment involves promotingangiogenesis- &/or vasculogenesis’ specifically includes (withoutlimitation) conditions such as wound healing, tissue and organregeneration or development, vasculodegenerative processes (e.g.critical limb- or brain ischemia, ischemic heart disease), embryonicdevelopment, and reproductive processes, e.g. female reproductionprocesses, such as follicle development in the corpus luteum duringovulation and placental growth during pregnancy.

As used herein, the term “tumor” refers to a malignant new growth thatarises from epithelium, found in skin or, more commonly, the lining ofbody organs, for example, breast, prostate, lung, kidney, pancreas,stomach or bowel. A tumor may also infiltrate into adjacent tissue andspread (metastasise) to distant organs, for example to bone, liver, lungor the brain. As used herein the term tumor includes both primary andmetastatic tumor cell types, such as but not limited to, melanoma,lymphoma, leukaemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma andtypes of tissue carcinoma, such as but not limited to, colorectalcancer, prostate cancer, small cell lung cancer and non-small cell lungcancer, breast cancer, pancreatic cancer, bladder cancer, renal cancer,gastric cancer, gliobastoma, primary liver cancer and ovarian cancers.

5.2 Detailed Description of the Invention

Angiogenesis is required for a number of physiological processes rangingfrom wound healing, tissue and organ regeneration, embryonic developmentand reproductive processes such as follicle development in the corpusluteum during ovulation and placental formation during pregnancy.Abnormal proliferation of blood vessels is an important component of avariety of diseases such as rheumatoid arthritis, retinopathies, andpsoriasis, these diseases (and related conditions) are referred toherein as “conditions where treatment involves inhibiting angiogenesisand/or vasculogenesis”. Angiogenesis is also an important factor in thegrowth and metastatic activity of solid tumors that rely onvascularization. Therefore, inhibitors of angiogenesis may be usedtherapeutically for the treatment of diseases resulting from oraccompanied by abnormal growth of blood vessels and for treatments ofmalignancies involving growth and spread of solid tumors.

The present invention relates to the use of Rspondin polypeptides,Rspondin nucleic acids and regulators or effectors or modulators ofRspondin polypeptides or Rspondin nucleic acids.

A first aspect of the present invention relates to the use of anRspondin polypeptide, an Rspondin nucleic acid or an Rspondin agonistfor the manufacture of an angiogenesis and/or vasculogenesis-promotingmedicament.

A further aspect of the invention relates to the use of an Rspondinantagonist for the manufacture of an angiogenesis and/orvasculogenesis-inhibiting medicament.

A further aspect of the invention refers to methods and reagents for thediagnosis or monitoring of angiogenesis- and/orvasculogenesis-associated processes, conditions or disorders, comprisingdetermining the amount, activity and/or expression of an Rspondinpolypeptide or an Rspondin nucleic acid in a sample. In a particularembodiment of the present invention, the amount, activity and/orexpression of an Rspondin polypeptide or an Rspondin nucleic acid insaid sample is compared to the amount, activity and/or expression ofsaid Rspondin polypeptide or Rspondin nucleic acid in a control sample.

Still a further aspect of the invention refers to recombinant cells andtransgenic non-human animals exhibiting modified, e.g. increased ordecreased Rspondin polypeptide expression.

Another aspect of the invention relates to the use of Rspondinpolypeptides, Rspondin nucleic acids, cells and transgenic non-humananimals to evaluate and screen test compounds for their ability tomodulate, e.g. stimulate or inhibit angiogenesis- and/orvasculogenesis-associated processes, conditions or disorders. Suchregulators of Rspondins may be used therapeutically. For example,agonists of Rspondins, e.g. Rspo2 and/or Rspo3 may be used in processessuch as wound healing; in contrast, antagonists of Rspo3 may be used inthe treatment of tumors that rely on vascularization for growth.

The invention is based, in part, on results from in situ-hybridizationindicating that Rspo3 is expressed in the embryonic vasculature. Theinvention is also based on the discovery that expression of Rspo3promotes endothelial cell differentiation, proliferation andmorphogenesis, while inhibition by antisense molecules in Xenopusembryos or targeted mutagenesis in knock out mice interferes withangiogenesis. The invention is also based on the discovery that Rspo3 isa positive regulator, which is both necessary and sufficient forexpression of the key angiogenic factor VEGF.

Accordingly, inhibition of rspondin molecules may be useful fortreatment of diseases resulting from abnormal proliferation of bloodvessels mediated by Rspondin, e.g. Rspo2 and/or Rspo3, and/or VEGF, inparticular in the treatment of conditions where treatment involvesinhibiting angiogenesis and/or vasculogenesis

The present invention relates to Rspondin polypeptides, Rspondin nucleicacids or regulators or effectors or modulators of Rspondin.

According to the present invention, an Rspondin polypeptide or aRspondin nucleic acid may be used for promoting angiogenesis and/orvasculogenesis, particularly for the manufacture of an angiogenesis-and/or vaculogenesis-promoting medicament.

This embodiment encompasses the prevention or treatment of a conditionwhere treatment involves promoting angiogenesis and/or vasculogenesis.

Rspondin polypeptides or Rspondin nucleic acids may be used in human orveterinary medicine, either alone or in combination with a furthermedicament, e.g. a further angiogenesis- and/or vasculogenesis-promotingmedicament such as a FGF, VEGF, PDGF, TNF or L-lysine.

A further aspect of this embodiment of the invention refers to a methodfor promoting angiogenesis in a cell or an organism comprisingincreasing the level, activity and/or expression of an Rspondinpolypeptide. This method may be carried out in vitro or in vivo, e.g.for therapeutic applications.

Further, this embodiment of the invention encompasses a method forpromoting angiogenesis comprising administering to a subject in needthereof a therapeutically effective dose of an Rspondin polypeptide or aRspondin nucleic acid, wherein the subject is preferably human.

A different embodiment of the present invention refers to the use of anRspondin antagonist for the manufacture of an angiogenesis- and/orvasculogenesis-inhibiting medicament. The Rspondin antagonist ispreferably an Rspondin2 and/or Rspondin3 antagonist.

This embodiment of the present invention encompasses the prevention ortreatment of a condition where treatment involves inhibitingangiogenesis and/or vasculogenesis.

In this embodiment, the Rspondin antagonist may be used in human orveterinary medicine, alone or in combination with a further medicament.For example, the treatment of tumors may comprise the combined use of anRspondin antagonist and an anti-tumor agent, e.g. a chemotherapeuticagent or an anti-tumor antibody, e.g. Bevacizumab, Endostatin,Thalidomide, Combrestatin A4, an anti VEGF antibody, SU 5416 or SU 6668.

Preferably, the nucleic acid molecules are recombinant DNA moleculesthat direct the recombinant expression of Rspondin polypeptides inappropriate host cells. Alternatively, nucleotide sequences whichhybridize to portions of an Rspondin-coding sequence may also be used innucleic acid amplification and/or hybridization assays, e.g. PCR,Southern and Northern blot analyses, etc.

Due to the inherent degeneracy of the genetic code, nucleic acidmolecules which encode substantially the same or a functionallyequivalent polypeptide, may be used in the practice of the invention forthe cloning and expression of an Rspondin, e.g. Rspo2 or 3 protein. SuchDNA sequences include those which are capable of hybridizing to theXenopus, and murine and/or human Rspondin sequences under stringentconditions. Preferably, hybridization under stringent conditions meansthat after washing for 1 h with 1 x SSC buffer and 0.1% SDS, preferablyat 55° C., more preferably at 62° C., and most preferably at 68° C.,particularly for 1 h in 0.2×SSC buffer and 0.1 SDS at 50° C., preferablyat 55° C., more preferably at 62° C. and most preferably at 68° C., apositive hybridization signal is observed.

The nucleic acid molecules of the invention may be engineered in orderto alter the Rspondin-coding sequence for a variety of purposesincluding but not limited to alterations which modify processing and/orexpression of the gene product. For example, mutations may be introducedusing techniques which are well known in the art, e.g. site-directedmutagenesis, to insert new restriction sites, to alter glycosylationpatterns, phosphorylation, etc. For example, in certain expressionsystems such as yeast, host cells may over glycosylate the gene product.When using such expression systems it may be preferable to alter theRspo2 or 3-coding sequence to eliminate any N-linked glycosylation site.

In another embodiment of the invention, the Rspondin nucleic acidsequence may be ligated to a heterologous sequence to encode a fusionprotein. For example, for screening of peptide libraries it may beuseful to encode a chimeric Rspondin protein expressing a heterologousepitope that is recognized by a commercially available antibody. Afusion protein may also be engineered to contain a cleavage site locatedbetween the Rspondin sequence and the heterologous protein sequence, sothat the Rspondin portion can be cleaved away from the heterologousmoiety.

In an alternative embodiment of the invention, the coding nucleic acidsequence can be synthesized in whole or in part, using chemical methodswell known in the art. See, for example, Caruthers, et al., 1980, Nuc.Acids Res. Symp. Ser. 7: 215-233; Crea and Horn, 180, Nuc. Acids Res.9(10): 2331; Matteucci and Caruthers, 1980, Tetrahedron Letters 21: 719;and Chow and Kempe, 1981, Nuc. Acids Res. 9(12): 2807-2817.Alternatively, the protein itself can be produced using chemical methodsto synthesize the Rspondin amino acid sequence in whole or in part. Forexample, peptides can be synthesized by solid phase techniques, cleavedfrom the resin, and purified by preparative high performance liquidchromatography (e.g., see Creighton, 1983, Proteins Structures AndMolecular Principles, W. H. Freeman and Co., N.Y. pp. 50-60). Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure; seeCreighton, 1983, Proteins, Structures and Molecular Principles, W. H.Freeman and Co., N.Y., pp. 34-49).

In order to express a biologically active Rspondin polypeptide, thenucleotide sequence coding for said polypeptide is inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. The Rspo gene products as well as host cells or celllines transfected or transformed with recombinant expression vectors canbe used for a variety of purposes. These include but are not limited togenerating antibodies (i.e., monoclonal or polyclonal) that bind toRspondin, including those that “neutralize” the activity of Rspondin.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the Rspondin-coding sequence andappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. See, for example, thetechniques described in Maniatis et al., 1989, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel etal., 1989, Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, N.Y.

A variety of host-expression vector systems may be utilized to expressthe Rspondin-coding sequence. These include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining the Rspondin-coding sequence; yeast celly transformed withrecombinant yeast expression vectors containing the Rspondin-codingsequence; insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus) containing the Rspo2 or 3-coding sequence;plant cell systems infected with recombinant virus expression vectors(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the Rspondin-coding sequence; or animal cell systemsinfected with recombinant virus expression vectors (e.g., adenovirus,vaccinia virus) including cell lines engineered to contain multiplecopies of the Rspondin DNA either stably amplified (CHO/dhfr-) orunstably amplified in double-minute chromosomes (e.g., murine celllines).

In an embodiment of the invention, Rspondin polypeptides, e.g. Rspo2and/or Rspo3, Respondin nucleic acids, and/or cell lines or non-humantransgenic animals that express an Rspondin may be used to screen forregulators or effectors or modulators of Rspondin that act as agonistsor antagonists of angiogenesis or vasculogenesis. For example,antibodies capable of neutralizing the activity of Rspondin, e.g. Rspo3in an endothelial proliferation assay, a chicken CAM assay and/or aXenopus VMZ differentiation assay, may be used to inhibit Rspondinfunction. Additionally, anti-Rspo3 antibodies which mimic VEGF activitymay be selected for pro-angiogenic applications, e.g. in wound healing.Alternatively, screening of peptide libraries or organic compounds withrecombinantly expressed soluble Rspondin polypeptides or cell lines ortransgenic non-human animals expressing an Rspondin polypeptide may beuseful for identification of therapeutic molecules that function bymodulating, e.g. inhibiting the biological activity of Rspondin and thusare suitable as angiogenesis and/or vasculogenesis regulators oreffectors or modulators of Rspondin, e.g. antagonists of Rspondin.

In an embodiment of the invention, engineered cell lines and/ortransgenic non-human animals which exhibit modified Rspondin expression,e.g. an increased or decreased expression of an Rspondin compared towild-type cell lines or animals, may be utilized to screen and identifyantagonists as well as agonists. Synthetic compounds, natural products,and other sources of potentially biologically active materials can bescreened in a number of ways to identify regulators or effectors ormodulators of Rspondin. The ability of a test compound to inhibit theactivity of an Rspondin polypeptide may be measured using an endothelialproliferation assay, a chicken CAM assay and/or a Xenopus VMZdifferentiation assay, such as those described in the Examples.

Identification of molecules that are able to bind to an Rspondinpolypeptide may be accomplished by screening a compound, e.g. a peptidelibrary with a recombinant soluble Rspondin polypeptide. To identify andisolate a compound that interacts and forms a complex with Rspondin, itis preferred to label or “tag” the Rspondin polypeptide. The Rspondinpolypeptide may be conjugated to labelling groups, e.g. enzymes such asalkaline phosphatase or horseradish peroxidase or to other reagents suchas fluorescent labels which may include fluorescein isothyiocynate(FITC), phycoerythrin (PE) or rhodamine. Conjugation of any given labelto Rspondin may be performed using techniques that are routine in theart. polypeptide containing an epitope for which a commerciallyavailable antibody exists. The epitope-specific antibody may be taggedusing methods well known in the art including labeling with enzymes,fluorescent dyes or colored or magnetic beads.

The “tagged” Rspondin polypeptide conjugate may be incubated with alibrary of immobilized compounds under suitable conditions, e.g. for 30minutes to one hour at 22° C. to allow complex formation between theRspondin polypeptide and an individual compound within the library. Thelibrary is then washed to remove any unbound Rspondin polypeptide. IfRspondin has been conjugated to alkaline phosphatase or horseradishperoxidase, the whole library may be poured into a petri dish containinga substrates for either alkaline phosphatase or peroxidase, for example,5-bromo-4-chloro-3-indoyl phosphate (BCIP) or 3,3′,4,4″-diamnobenzidine(DAB), respectively. After incubating for several minutes, thecompound/solid phase-Rspondin complex changes color, and can be easilyidentified and isolated physically under a dissecting microscope with amicromanipulator. If a fluorescent tagged Rspondin molecule has beenused, complexes may be isolated by fluorescent activated sorting. If achimeric Rspondin polypeptide expressing a heterologous epitope has beenused, detection of the compound/Rspondin complex may be accomplished byusing a labeled epitope-specific antibody. Once isolated, the identityof the compound attached to the solid phase support may be determined,e.g. by peptide sequencing.

Cell lines or non-human transgenic animals that express Rspondin, e.g.Rspondin3, may be used to screen for regulators or effectors ormodulators of Rspondin in a number of ways.

The ability of a regulator or effector or modulator of Rspondin tointerfere with Rspondin activity and/or Rspondin signal transduction maybe measured using an endothelial proliferation assay, a chicken CAMassay or a Xenopus VMZ differentiation assay. Other responses such asactivation or suppression of catalytic activity, phosphorylation ordephosphorylation of other proteins, activation or modulation of secondmessenger production, changes in cellular ion levels, association,dissociation or translocation of signalling molecules, or transcriptionor translation of specific genes may also be monitored. These assays maybe performed using conventional techniques developed for these purposesin the course of screening.

Ligand binding to its cellular receptor may, via signal transductionpathways, affect a variety of cellular processes. Cellular processesunder the control of the Rspondin signaling pathway may include, but arenot limited to, normal cellular functions, proliferation,differentiation, maintenance of cell shape, and adhesion, in addition toabnormal or potentially deleterious processes such as unregulated cellproliferation, loss of contact inhibition, blocking of differentiationor cell death. The qualitative or quantitative observation andmeasurement of any of the described cellular processes by techniquesknown in the art may be advantageously used as a means of scoring forsignal transduction in the course of screening.

Various embodiments are described below for screening, identificationand evaluation of compounds that interact with Rspondin, which compoundsmay affect various cellular processes under the control of the Rspondinsignalling pathway.

The present invention includes a method for identifying a regulator,effector or modulator of Rspondin, comprising:

-   (a) contacting the putative regulator, effector or modulator of    Rspondin with an Rspondin polypeptide, in pure or semi-pure form, or    in a whole live or fixed cell or in a non-human transgenic animal,-   (b) measuring the effect of the putative regulator, effector or    modulator of Rspondin on the Rspondin polypeptide, the activity of    the Rspondin, and/or on a phenotypic property of the cell or the    organism mediated by the Rspondin,-   (c) comparing the measured effect to that without the putative    regulator, effector or modulator of Rspondin, thereby determining    whether the putative regulator, effector or modulator of Rspondin    stimulates or inhibits the intracellular response of the Rspondin.

Rspondins, e.g. Rspo3, useful in identifying a regulator, effector ormodulator of Rspondin may be functionally equivalent to Rspondin. Afunctional equivalent to Rspondin may be prepared from a naturallyoccurring or recombinantly expressed Rspondin by proteolytic cleavagefollowed by conventional purification procedures known to those skilledin the art. Alternatively, the functional derivative may be produced byrecombinant DNA technology by expressing parts of Rspondin which includethe functional domain in suitable cells. Functional derivatives may alsobe chemically synthesized. Cells expressing Rspo3 may be used as asource of Rspondin, crude or purified, for testing in these assays.Alternatively, whole live or fixed cells may be used directly in thoseassays.

Rspondin signal transduction activity may be measured by an endothelialproliferation assay, a chicken CAM assay or a Xenopus VMZdifferentiation assay and/or by monitoring the cellular processescontrolled by the signal.

The invention also includes a method whereby a molecule capable ofbinding to an Rspondin polypeptide may be identified comprising:

-   (a) immobilizing an Rspondin polypeptide or a functional equivalent    thereof to a solid phase matrix;-   (b) contacting the molecule with the solid phase matrix produced in    step (a), for an interval sufficient to allow the molecule to bind;-   (c) washing away any unbound material from the solid phase matrix;-   (d) detecting the presence of the molecule bound to the solid phase.

The above method may further include the step of:

-   (e) eluting the bound molecule from the solid phase matrix, thereby    isolating the molecule.

The above method may further include the step of:

-   (f) identifying the molecule eluted.

Various procedures known in the art may be used for the production ofantibodies to epitopes of an Rspondin polypeptide, e.g. Rpo2 or Rspo3.

Monoclonal antibodies that bind to an Rspondin polypeptide may beradioactively labelled allowing one to follow their location anddistribution in the body after injection. Radioactivity taggedantibodies may be used as a non-invasive diagnostic tool for imaging denovo vascularization associated with conditions where treatment involvesinhibiting angiogenesis and/or vasculogenesis.

Immunotoxins may also be designed which target cytotoxic agents tospecific sites in the body. For example, high affinity Rspondin-specificmonoclonal antibodies may be covalently complexed to bacterial or planttoxins, such as diptheria toxin, abrin or ricin. A general method ofpreparation of antibody/hybrid molecules may involve use ofthiol-crosslinking reagents such as SPDP, which attack the primary aminogroups on the antibody and by disulfide exchange, attach the toxin tothe antibody. The hybrid antibodies may be used to specificallyeliminate Rspondin expressing endothelial cells.

For the production of antibodies, various host animals may be immunizedby injection with the Rspondin polypeptide including but not limited torabbits, mice, rats, etc. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Monoclonal antibodies to Rspondin may be prepared by using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include but are not limited to thehybridoma technique originally described by Kohler and Milstein,(Nature, 1975, 256: 495-497), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today, 4: 72; Cote et al., 1983, Proc.Natl. Acad. Sci., 80: 2026-2030) and the EBV-hybridoma technique (Coleet al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). In addition, techniques developed for the productionof “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81: 6851-6855; Neuberger et al., 1984, Nature, 312: 604-608; Takeda etal., 1985, Nature, 314: 452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produceRspondin-specific single chain antibodies.

Antibody fragments which contain specific binding sites for Rspo3 may begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246: 1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityto Rspondin.

Antibodies to Rspondin polypeptides may antagonise the activity ofRspondin by preventing it from binding to its usual partner in the Wntsignalling cascade. Therefore, antibodies which bind specifically toRspondin, in particular to Rspo2 or Rspo3, may be antagonists ofRspondin which may be used to inhibit angiogenesis and/orvasculogenesis.

In addition, mutated forms of Rspondin, having a dominant negativeeffect, may be expressed in targeted cell populations to inhibit theactivity of endogenously expressed wild-type Rspo3.

Included in the scope of the invention are nucleic acid antagonists ofRspondin. Anti-sense RNA and DNA molecules act to directly block thetranslation of mRNA by binding to targeted mRNA and preventing proteintranslation. In regard to antisense DNA, oligodeoxyribonucleotidesderived from the translation initiation site, e.g., between −10 and +10regions of the Rspondin nucleotide sequence, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by a endonucleolytic cleavage. Withinthe scope of the invention are engineered hammerhead motif ribozymemolecules that specifically and efficiently catalyze endonucleolyticcleavage of Rspo3 RNA sequences.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for predicted structuralfeatures such as secondary structure that may render the oligonucleotidesequence unsuitable. The suitability of candidate targets may also beevaluated by testing their accessibility to hybridization withcomplementary oligonucleotides, using ribonuclease protection assays.

RNAi molecules are double-stranded RNA molecules or analogues thereofcapable of mediating RNA interference of a target mRNA molecule, e.g.siRNA molecules which are short double-stranded RNA molecules with alength of preferably 19-25 nucleotides and optionally at least one3′-overhang or precursors thereof or DNA molecules coding therefor.Anti-sense RNA and DNA molecules, ribozymes and RNAi molecules of theinvention may be prepared by any method known in the art for thesynthesis of RNA molecules. These include techniques for chemicallysynthesizing oligodeoxyribonucleotides well known in the art such as forexample solid phase phosphoramidite chemical synthesis. Alternatively,RNA molecules may be generated by in vitro and in vivo transcription ofDNA sequences encoding the antisense RNA molecule. Such DNA sequencesmay be incorporated into a wide variety of vectors which incorporatesuitable RNA polymerase promoters such as the T7 or SP6 polymerasepromoters. Alternatively, antisense cDNA constructs that synthesizeantisense RNA constitutively or inducibly, depending on the promoterused, can be introduced stably into cell lines.

Various modifications to the DNA molecules may be introduced as a meansof increasing intracellular stability and half-life. Possiblemodifications include but are not limited to the addition of flankingsequences of Morpholino derivatives as well as ribo- ordeoxy-nucleotides to the 5′ and/or 3′ ends of the molecule or the use ofphosphorothioate or 2′ O-methyl rather than phosphodiesterase linkageswithin the oligodeoxyribonucleotide backbone.

Expression and functional activity of Rspo3 correlates with thedevelopment of the vasculature and endothelial cell proliferation,indicating that Rspo3 is involved in the vascularization process.Rspondins, such as Rspo2 or 3, induce VEGF, and VEGF has been shown tobe a mitogenic growth factor known to act exclusively on endothelialcells (Ferrara, N. and Henzel, W. J., 1989, Biochem. Biophys. Res. Comm.161: 851-858).

In one embodiment of the invention, Rspondin polypeptides such as Rspo2or 3, can be administered in vivo to modulate angiogenesis and/orvasculogenesis. For example, the administration of Rspo2 or 3 may beused to treat conditions where treatment involves promoting angiogenesisand/or vasculogenesis, whereas antagonists of Rspo2 or 3 may be used totreat conditions where treatment involves inhibiting angiogenesis and/orvasculogenesis.

In a particular embodiment of the invention, Rspondin agonists may beused to treat conditions wherein treatment involves promotingangiogenesis and/or vasculogenesis. In a particular embodiment saidconditions are selected from the group consisting of wound healing,tissue and organ regeneration or development, vasculodegenerativeprocesses (e.g. critical limb- or brain ischemia, ischemic heartdisease), embryonic development, and reproductive processes such asfollicle development in the corpus luteum during ovulation and placentalgrowth during pregnancy. In a further particular embodiment saidcondition is selected from wound healing, tissue and organ regenerationor development, vasculodegenerative processes (e.g. critical limb- orbrain ischemia, ischemic heart disease).

In a particular embodiment of the invention the Rspondin agonist is anRspo2 agonist or an Rspo3 agonist.

In a particular embodiment of the invention the Rspondin agonist isselected from an Rspondin polypeptide, an Rspondin nucleic acid or asmall molecule. In a most particular embodiment of the invention anRspondin polypeptide may be used to treat conditions wherein treatmentinvolves promoting angiogenesis or vasculogenesis. In a most particularembodiment of the invention an Rspondin nucleic acid may be used totreat conditions wherein treatment involves promoting angiogenesisand/or vasculogenesis.

In a particular embodiment of the invention Rspondin antagonists may beused in the treatment of conditions where treatment involves inhibitingangiogenesis e.g. tumor growth and metastatic activity, atherosclerosis,stenosis, restenosis, retinopathy, macular degeneration, psoriasis andrheumatoid arthritis. In a particular embodiment said condition is solidtumor growth. In a further particular embodiment said condition ismacular degeneration. In a further particular embodiment said conditionis rheumatoid arthritis.

In a particular embodiment of the invention the Rspondin antagonist isan Rspo2 antagonist or an Rspo3 antagonist.

In a particular embodiment of the invention the Rspondin antagonist isselected from an Rspondin antibody or a nucleic acid capable ofinhibiting Rspondin translation, transcription, expression and/oractivity. In a most particular embodiment of the invention an Rspondinantibody may be used to treat conditions wherein treatment involvesinhibiting angiogenesis or vasculogenesis. In a most particularembodiment of the invention a nucleic acid capable of inhibitingRspondin translation, transcription, expression and/or activity may beused to treat conditions wherein treatment involves promotingangiogenesis or vasculogenesis. In a most particular embodiment of theinvention an siRNA or antisense nucleic acid against Rspondin may beused to treat conditions where treatment involves promoting angiogenesisor vasculogenesis.

Pharmaceutically active regulators or effectors or modulators ofRspondin can be administered to a patient either by itself, or inpharmaceutical compositions where it is mixed with suitable carriers orexcipient(s).

Depending on the specific conditions being treated, these agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in “Remington'sPharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latestedition. Suitable routes may, for example, include oral, rectal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, or, in the caseof solid tumors, directly injected into a solid tumor. For injection,the agents of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

The regulators or effectors or modulators of Rspondin can be formulatedreadily using pharmaceutically acceptable carriers well known in the artinto dosages suitable for oral administration. Such carriers enable theregulators or effectors or modulators of Rspondin of the invention to beformulated as tablets, pills, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a patient to be treated.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the regulators or effectors or modulatorsof Rspondin are contained in an effective amount to achieve its intendedpurpose. Determination of the effective amounts is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

In addition to the regulators or effectors or modulators of Rspondinthese pharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the regulators or effectors or modulators ofRspondin into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the regulators or effectors or modulators ofRspondin in water-soluble form. Additionally, suspensions of theregulators or effectors or modulators of Rspondin may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of theregulators or effectors or modulators of Rspondin to allow for thepreparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe regulators or effectors or modulators of Rspondin with solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of regulators or effectors or modulators ofRspondin doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the regulators or effectors or modulators ofRspondin in admixture with filler such as lactose, binders such asstarches, and/or lubricants such as talc or magnesium stearate and,optionally, stabilizers. In soft capsules, the regulators or effectorsor modulators of Rspondin may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

Compositions comprising a regulators or effectors or modulators ofRspondin of the invention formulated in a compatible pharmaceuticalcarrier may be prepared, placed in an appropriate container, andlabelled for treatment of an indicated condition. Suitable conditionsindicated on the label may include treatment of a tumor, such as aglioma or glioblastoma; and. other conditions where treatment involvesinhibiting angiogenesis and/or vasculogenesis.

Compositions comprising a regulators or effectors or modulators ofRspondin of the invention formulated in a compatible pharmaceuticalcarrier may be prepared, placed in an appropriate container, andlabelled for treatment of an indicated condition. Suitable conditionsindicated on the label may include treatment of a conditions wheretreatment involves promoting angiogenesis and/or vasculogenesis, inparticular wound healing.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Many of the regulators or effectors or modulators of Rspondin of theinvention may be provided as salts with pharmaceutically compatiblecounterions. Pharmaceutically compatible salts may be formed with manyacids, including but not limited to hydrochloric, sulfuric, acetic,lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble inaqueous or other protonic solvents that are the corresponding free baseforms.

For any regulator or effector or modulator of Rspondin used in themethod of the invention, the therapeutically effective dose can beestimated initially from cell culture assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC50 as determined in cell culture (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of the PTP activity). Such information can be used to moreaccurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the regulatoror effector or modulator of Rspondin that results in amelioration ofsymptoms or a prolongation of survival in a patient. Toxicity andtherapeutic efficacy of such regulators or effectors or modulators ofRspondin can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., for determining the LD50 (thedose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ ED50. Regulators or effectors ormodulators of Rspondin which exhibit large therapeutic indices arepreferred. The data obtained from these cell culture assays and animalstudies can be used in formulating a range of dosage for use in human.The dosage of such regulators or effectors or modulators of Rspondinlies preferably within a range of circulating concentrations thatinclude the ED50 with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition. (See e.g. Fingl et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the regulators or effectors or modulators of Rspondinwhich are sufficient to maintain the Rspo3 inhibitory or promotingeffects. Usual patient dosages for systemic administration range from1-2000 mg/day, commonly from 1-250 mg/day, and typically from 10-150mg/day. Stated in terms of patient body weight, usual dosages range from0.02-25 mg/kg/day, commonly from 0.02-3 mg/kg/day, typically from0.2-1.5 mg/kg/day. Stated in terms of patient body surface areas, usualdosages range from 0.5-1200 mg/m² /day, commonly from 0.5-150 mg/m²/day,typically from 5-100 mg/m²/day.

Dosage amount and interval may be adjusted individually to provideplasma levels of the regulator or effector or modulator of Rspondinwhich are sufficient to maintain the Rspondin inhibitory or promotingeffects. Usual average plasma levels should be maintained within 50-5000μg/ml, commonly 50-1000 μg/ml, and typically 100-500 μg/ml.

Alternately, one may administer the regulator or effector or modulatorof Rspondin in a local rather than systemic manner, for example, viainjection of the regulator or effector or modulator of Rspondin directlyinto a tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the pharmaceutical composition in atargeted drug delivery system, for example, in a liposome coated withtumor-specific antibody. The liposomes will be targeted to and taken upselectively by the tumor.

In cases of local administration or selective uptake, the effectivelocal concentration of the pharmaceutical composition may not be relatedto plasma concentration.

The Rspondin nucleic acids or compounds capable of binding to Rspondin,such as antibodies may be used for diagnostic purposes for detection ofRspondin expression in angiogenesis- and/or vasculogenesis-associatedprocesses, conditions or disorders.

Reagents suitable for detecting Rspondins, such as Rspondin nucleicacids or compounds capable of binding to Respondin may have a number ofuses for the diagnosis of processes, conditions or diseases resultingfrom, associated with and/or accompanied by, aberrant expression ofRspondin. The diagnostic procedures are preferably carried out onsamples obtained from a subject, e.g. a human patient, e.g. samples frombody fluids such as whole blood, plasma, serum or urine, or tissuesamples such as biopsy or autopsy samples. For example, the Rspondinsequence may be used in amplification, e.g. hybridization assays todiagnose abnormalities of Rspondin expression; e.g., Southern orNorthern analysis, including in situ hybridization assays.

The Rspondin cDNA may be used as a probe to detect the expression of thecorresponding mRNA. In a specific example described herein, theexpression of Rspo3 mRNA in mouse embryos was analyzed (FIG. 4). Rspo3mRNA was found to be enriched in embryonic vessels, indicating a rolefor Rspo3 in endothelial cell proliferation.

Further, the present invention is explained in more detail by thefollowing Example.

6 EXAMPLE

6.1 Materials and Methods

Rspo Coding Sequences

The nucleotide-coding sequence and deduced amino acid sequence of themurine and Xenopus Rspondin genes as deposited in Genbank used here are

-   -   X. laevis Rspondin 2 [gi:54145367] (SEQ ID NO: 1)    -   X. tropicalis Rspondin 3 [gi:114149217] (SEQ ID NO: 2)    -   M. musculus Rspondin 3 [NM_028351] (SEQ ID NO: 3)        Mouse and Xenopus Embryos

Balb/c mice were mated overnight and the morning of vaginal plugdetection was defined as 1/2 day of gestation. For routine histologicalanalysis, tissues were fixed in 4% paraformaldehyde overnight andembedded in paraffin wax for sectioning. Generally, 4 μm sections werecut and stained with Hemalum and Eosine. For wholemount in situhybridization, the embryos were fixed and processed as described (delBarco et al., 2003, Genes Dev. 17, 2239-2244). Xenopus embryos wereobtained by in vitro fertilization and cultivated as described (Gawantkaet al 1995 EMBO J. 14, 6268-79). Xenopus embryos were fixed andprocessed for whole mount in situ hybridization as described (Bradley etal., 1996 Development 122, 2739-2750). Ventral marginal zone wereexcised and cultivated as described (Gawantka et al 1995 EMBO J. 14,6268-79). Full length Rspo3 cDNAs were used to generate antisenseriboprobes.

Rspo3 knock out mice were obtained by targeted mutagenesis of murineRspo3 (gi:94388197) in mouse embryonic stem cells following standardprocedures, using a targeting vector shown in FIG. 5. Transgenic micewere generated on a C57BL/6 background via standard diploid injection.Homozygous mutant embryos were generated by heterozygote intercrosses.C57BL/6 heterozygotes were then backcrossed to CD1 females for at least6 generations. No serious phenotypic differences were detected betweenhomozygous embryos in C57BL/6 and CD1 background. Mouse tail tips orportions of yolk sacs or embryos were used for genotyping by PCR.Genotyping was routinely performed by PCR analysis using 3 primers,5′-ATGCTTTGAGGCTTGTGACC-3′ (SEQ ID NO: 4), 5′-TGCACCGACTCCAGTACTGG-3′(SEQ ID NO: 5) and 5′-TACATTCTGGTTTCTCATCTGG-3′ (SEQ ID NO: 6).

RT-PCR

RT-PCR assays were carried out as described (Gawantka et al 1995 EMBO J.14,6268-79); additional primers were; XSCL (forward,actcaccctccagacaagaa (SEQ ID NO: 7); reverse, atttaatcaccgctgcccac (SEQID NO: 8)); α-globin (forward, tccctcagaccaaaacctac (SEQ ID NO: 9);reverse, cccctcaattttatgctggac (SEQ ID NO: 10)); Xmsr (forward,aacttcgctctcgctcctccatac (SEQ ID NO: 11); reverse,gccagcagatagcaaacaccac (SEQ ID NO: 12)), VEGF (forward,aggcgagggagaccataaac (SEQ ID NO: 13); reverse, tctgctgcattcacactgac (SEQID NO: 14)).

Preparation of Xenopus laevis Rspo2-conditioned Medium

Transfection of HEK293T cells with Xenopus laevis Rspo2 (gi:54145367)and harvest of conditioned medium were as described (Kazanskaya et al.,2004, Dev. Cell 7, 525-534).

Endothelial Proliferation Assay

Human Umbilical Vein Endothelial Cells (HUVEC) (PromoCell) were culturedin Endothelial cell Growth Medium (Promocell) supplemented with 10%fetal bovine serum (FBS). For proliferation studies, cells were platedat 50% confluence in 96-well plate, next day they were supplemented withVEGF and Xenopus laevis Rspo2 proteins for 48 h, after which BrdU (10μM) was added to each well for 4 h. BrdU analysis of cell proliferationwas carried out using Cell Proliferation ELISA BrdU chemiluminescentfrom Roche Applied Science.

Chorioallantoic Membrane (CAM) Assay

For chicken chorioallantoic membrane (CAM) assay, chicken eggs wereincubated at 37° C. in a humidified chamber. On day 3 of development, awindow was made in the outer shell and on 6 day of development a 20 μlof Rspo2 or control beads or filter disk (3 MM Whatman-8 mm diameter)carrying recombinant VEGF (Sigma-Aldrich, 100 ng/filter) was placed ontothe surface of the CAM. The beads (ANTI-FLAG M2-Agarose, Sigma) wereincubated overnight with Xenopus laevis Rspo2-conditioned medium orcontrol medium from untransfected HEK 293T cells and washed 3 times inPBS. After 5 days of incubation, the filter disks and the attached CAMwere excised, washed with PBS and processed for histology usingHematoxylin-Eosine staining.

Antisense Morpholino Oligonucleotide

Based on Xenopus tropicalis Rspo3 cDNA sequence (gi:114149217), anantisense morpholino oligonucleotide was designed (sequence: 5′:atgcaattgcgactgctttctctgt (SEQ ID NO: 15)).

6.2 Results

FIG. 1 shows that an antisense morpholino oligonucleotide which isdirected against Xenopus tropicalis Rspo3, inhibited the development offorming blood vessels in Xenopus tadpoles. A marker for forming bloodvessels is the gene msr, which was down-regulated. Inhibition of bloodvessel development—in other words embryonic angiogenesis—is accompaniedby expansion of blood cell development, since blood cell markersα-globin and SCL are expanded. The results suggest that Rspo3 is adevelopmental regulator that switches cell fate between blood and bloodvessel development. The specificity of the morpholino-induced phenotypefor inhibition of Rspo3 is demonstrated by the rescue experiment in FIG.2. In this experiment, the related molecule Rspo2 was able to revert theexpansion of blood marker α-globin.

The ability of Rspo2 to promote angiogenesis in Xenopus embryos is shownin an ventral marginal zone (VMZ) assay FIG. 3. Overexpression of Rspo2mRNA inhibits blood cell markers and induces the endothelial marker msr,as well as the angiogenic factor VEGF. Conversely, the requirement ofendogenous Rspo3 for embryonic angiogenesis is shown by the inhibitionof msr and VEGF by an antisense morpholino oligonucleotide.

By way of the examples in FIGS. 1-3 it is demonstrated that inhibitionof Rspo3 in a vertebrate inhibits VEGF, vasculogenesis and angiogenesis.Therefore, antagonists of Rspo3 will be useful to deliberately inhibitVEGF, vasculogenesis and angiogenesis where this is useful, e.g. inconditions where treatment involves inhibiting angiogenesis and/orvasculogenesis.

The ability of Rspondins to promote angiogenesis is not limited toXenopus but also extends to mammals, e.g. to the mouse. Rspo3 isexpressed in murine embryonic blood vessels (FIG. 4). Furthermore, Rspo3mutant mice show defective angiogenesis. This is demonstrated by theearly lethality of such mutant mice, which show internal bleedings, asis characteristic for a failure to form blood vessels (FIG. 6). Thedeficient angiogenesis is also evidenced by the reduced blood vessels inthe yolk sac of mutant embryos (FIG. 7). Furthermore, the inactivationof Rspo3 is accompanied with downregulation of VEGF in mutant placentas(FIG. 8). By way of these examples it is again demonstrated thatinhibition of Rspo3 in a mammal inhibits VEGF, vasculogenesis andangiogenesis.

The ability of Rspo2 to induce angiogenesis is demonstrated in twostandard in vitro angiogenesis assays. In the chicken choriallantoismembrane (CAM) assay, the ability of regulators or effectors ormodulators of Rspondin to promote the growth of endothelial cells andblood vessels is measured. A strong induction of endothelial cell growthand of blood vessels was observed following implantation of beads soakedwith VEGF or Rspo2 conditioned medium (FIG. 9). Furthermore, Rspo2conditioned medium induced branching morphogenesis in endothelial cells(FIG. 10), a characteristic response to angiogenic factors. In additionRspo2 induced proliferation of endothelial cells, similar to theangiogenic factor VEGF (FIG. 11).

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention, and any clones, DNA or functionally equivalents to Rspondinare within the scope of the invention. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description andaccompanying drawings. Such modifications are intended to fall withinthe scope of the appended claims.

All references cited herein are hereby incorporated by reference intheir entirety.

The invention claimed is:
 1. A method for inhibiting angiogenesis in asubject in need of such treatment comprising administering to thesubject a therapeutically effective dose of an Rspondin3 antagonist,wherein the Rspondin3 antagonist is a nucleic acid molecule capable ofinhibiting Rspondin3 translation, transcription, expression and/oractivity, and wherein the Rspondin3 antagonist is an antisense molecule.2. The method of claim 1 wherein the Rspondin3 antagonist is anantisense molecule capable of inhibiting transcription of Rspondin3nucleic acid encoding Rspondin3 polypeptide comprising the amino acidsequence set out in SEQ ID NO: 21 or comprising the sequence set out inSEQ ID NO: 20 or capable of inhibiting translation, expression and/oractivity of Rspondin3 polypeptide comprising the amino acid sequence setout in SEQ ID NO: 21.